Protein microarrays are important tools for the discovery of biomarkers, protein-protein interactions, and protein-glycan interactions (MacBeath, “Protein Microarrays and Proteomics,” Nat. Genetics 32:526-32 (2002); Krishnamoorthy et al., “Glycomic Analysis: An Array of Technologies,” ACS Chem. Biol. 4(9):715-32 (2009)). Lectin microarrays are protein microarrays for the detection and analysis of cellular glycosylation (Pilobello et al., “A Ratiometric Lectin Microarray Approach to Analysis of the Dynamic Mammalian Glycome,” Proc. Natl. Acad. Sci. U.S.A. 104(28):11534-9 (2007); Pilobello et al., “Development of a Lectin Microarray for the Rapid Analysis of Protein Glycopatterns,” Chembiochem 6(6):985-9 (2005)).
The current approach of creating lectin microarrays is limited, in part, by glycan-detecting sensitivity as a function of protein concentration. Recently, a method to utilize bacterial lectins for glycomic analysis has been developed (Hsu et al., “A Simple Strategy for the Creation of a Recombinant Lectin Microarray,” Mol. Biosyst. 4(6):654-62 (2008)). These lectins are recombinantly expressed as fusion proteins containing an N-terminal glutathione-S-transferase-(“GST”-) and a polyhistidine-(“His6”-) tag. Previous studies have shown that GST-fusion proteins can be oriented on a glutathione (“GSH”) surface. Orienting lectins using the GST-GSH interaction on glutathione-treated slides was found to have a significant (>2-fold) increase in their activity. Although this was beneficial for a recombinant lectin microarray, a solely recombinant array means a significant loss of diversity in binding proteins on an array. Thus, one drawback of this approach is the requirement that all proteins to be displayed on the array have a GST-moiety. This means that only recombinant proteins can be utilized on a single array. It would be useful to also employ lectins (or other proteins) from natural (non-recombinant) sources.
In one approach, two distinct surfaces were created in a single subarray, displaying the benefits of orienting recombinant lectins which increased the sensitivity of the lectins to detect in the nanomolar (nM) concentration of glycoprotein (Propheter et al., “Fabrication of an Oriented Lectin Microarray” Chembiochem. May 18, 2010). This, compared to other methods of detection (such as evanescent wave detection), proved simple and inexpensive. The creation of a dual-surface array was also cost-effective. However, available probes are limited to recombinant lectins currently available. In the search for a methodology for the inclusion of oriented lectins to the standard microarray in which a single slide surface chemistry is utilized, a practical approach that is neither tedious nor expensive and is widely applicable to similar systems is needed.
The activity of proteins in protein microarrays is always a concern. For example, the random coupling of proteins via amide coupling chemistry to lysine residues often impinges upon protein activity, causing occlusion of active sites and in some cases denaturation of the protein structure. Control over the orientation of proteins is an important factor in enhancing activity on a solid support. However, in arrays, often there is also a desire to maximize the number of probes to be utilized. Thus, the limitations inherent in a recombinant array, i.e., that every protein has to be expressed with the same tag to be bound to a uniform surface, is undesirable if arrays that include proteins from a wide variety of sources are desired. Creating surfaces that can accommodate both recombinant and untagged proteins would enhance the utility and activity of current protein arrays.
The present invention is directed to overcoming these and other limitations in the art.